Interaction between the HIV Rev and the cellular LEDGF/p75 proteins under in vitro and in vivo conditions. (A) An ELISA assay system was used to determine the Rev(-GFP)-LEDGF/p75, Rev(-GFP)-IN and IN-LEDGF/p75 interactions. LEDGF/p75 or IN were bound to ELISA plate and the binding of Rev(-GFP) to LEDGF/p75 () or to IN () as well as the binding of LEDGF/p75 to IN () was estimated as described in Materials and Methods. For control, the binding of the GFP to LEDGF/p75 () and to IN () were studied. (B) Apparent K_d as calculated from the results presented in (A). (C) The BiFC method was used in yeast cells to demonstrate Rev-LEDGF/p75 interactions. Cells were transfected with the indicated expression vectors and, following incubation appearance of fluorescence, due to protein complementation, was visualized by fluorescent confocal microscopy. The Linker plasmid contains only the half GFP (GN or GC) and a linker as described in Materials and Methods. The scale bar represents 1 μm. All other details as described in Materials and Methods.

Rev induced disruption of the IN-LEDGF/p75 complex. (A) The presence of Rev, IN, LEDGF/p75 and Actin (control) was detected by Western blots in cell lysates obtained from HIV-1-infected H9 lymphocytes at different times after infection using the appropriate antibodies. Rev, IN, LEDGF/p75 and Actin also were precipitated from the cytoplasm, nuclei and PIC fractions obtained from HIV-1-infected H9 lymphocytes at different times PI by the indicated specific antibodies. (B) Co-IP of Rev-IN and Rev-LEDGF/p75, but not IN-LEDGF/p75, can be observed in Rev10⁺ cells. (C) Only IN-LEDGF/p75 co-IP can be observed in cells infected with ΔRev HIV-1. (D) Only Rev-IN co-IP can be observed in LEDGF/p75-knockdown cells infected with WT HIV-1. (E) In LEDGF/p75-knockdown cells infected with ΔRev HIV-1, only IN can be detected. (F) Co-IP of Rev-IN, IN-LEDGF/p75 and Rev-LEDGF/p75, which is observed in infected cell lysates, is interrupted specifically by pretreating the cells with 150 μM of specific peptides that contain the interaction-mediating sequences. Quantitative estimation of the bands, performed by Image Gauge V3.46 software, is presented in supplementary Table 1A–F, respectively. All other details are described in Materials and Methods.

Summary of the results obtained on the Rev-LEDGF/p75 interaction and a proposed integration model. (A) A scheme illustrating the interacting sequences of IN (red), Rev (green) and LEDGF/p75 (blue), as was revealed mainly by the ELISA-based assay system, which mediate the binding between these proteins. (B) A table summarizing the abilities of the various IN-, Rev- and LEDGF/p75-derived peptides to specifically disrupt IN-LEDGF/p75, Rev-IN and Rev-LEDGF/p75 interactions. (C) A proposed model of the HIV-1 integration mechanism. IN (red), LEDGF/p75 (blue), Rev (green), viral DNA (purple) and host cell DNA (light blue). Following virus infection, LEDGF/p75 interacts with an IN (dimer)-DNA (I) complex resulting in the formation of a IN-LEDGF/p75 (tetramer)-DNA complex in the viral PIC (II) (47). Despite the presence of numerous IN-LEDGF/p75 (tetramer)-DNA complexes, only 1 to 2 copies of the viral DNA are integrated into the host chromosomal DNA (III). Most of the IN-LEDGF/p75 complexes are dissociated by increasing amounts of transcribed Rev, resulting in the formation of inhibitory Rev-IN-DNA complex (IV) and a Rev-LEDGF/p75 complex (V).

Rev promotes dissociation of the IN-LEDGF/p75 complex while the LEDGF/p75 fails to promote dissociation of the Rev-IN complex. (A) Rev-GFP conjugate () and Tat () were added to plate-bound IN-LEDGF/p75 complex and the amount of bound LEDGF/p75 was estimated. (B) LEDGF/p75 () and Tat () were added to plate-bound Rev(-GFP)-IN complex and the amount of bound Rev-GFP was estimated. (C) Same as in B but the amount of bound LEDGF/p75 and Tat was estimated. (D) Rev-GFP (), Rev-GFP and LEDGF/p75 () or Rev-GFP and Tat () were added to plate-bound IN and the amount of bound Rev-GFP was estimated. (E) LEDGF/p75 (), Rev-GFP and LEDGF/p75 () or LEDGF/p75 and Tat () were added to plate-bound IN and the amount of bound LEDGF/p75 was estimated. (F) Apparent K_d as calculated from the changes in affinity presented in (D) and (E). All other details are described in Materials and Methods.

The effect of the combination of LEDGF/p75 and Rev on IN enzymatic activity in vitro and in LEDGF/p75-knockdown cells. (A) IN (390 nM) was added to the substrate DNA and then either LEDGF/p75, Rev-GFP or both, in the order indicated by arrows, were added, or, as control, none of them were added (no addition) and IN enzymatic activity was determined as described in Materials and Methods. (B) An ELISA system was used to determine binding of biotinilated DNA to plate-bound IN (), IN + LEDGF/p75 (), IN + Rev-GFP (), IN + Rev-GFP + LEDGF/p75 (), and IN + GFP () used as a control. Binding was estimated as described in Materials and methods. (C) Apparent K_d as calculated for the results presented in (B). (D) Hela P4 (), LEDGF/p75-knockdown () and LEDGF/p75-knockdown-Rev-expressing () cells were infected by the indicated viruses (at a MOI of 1) and integration events per cell were determined at 24 h PI. (E) LEDGF/p75-knockdown cells were infected with ΔRev HIV-1, and IN (red), DNA (blue), Rev (green) and LEDGF/p75 (gray) were immunostained as described in Materials and Methods. Scale bar represents 10 μm. (F) Same as (D) except that p24 production was determined at 72 h PI. (G) Same as (D) except that viral cDNA was determined at 12 h PI. AZT was added at 2 μM.